This application seeks to develop and apply new small molecule strategies that go beyond fluorescent proteins to image protein function and discrete RNAs in vitro or inside the cell and create tools that impact discovery in biology and medicine. One tool is based on bipartite tetracysteine (C4) display, in which the linear C4 binding site for a biarsenical dye is split between two approximated regions of a folded protein or two members of a protein partnership. During the first funding period we reported that the linear tetracysteine (C4) sequence preferred by biarsenicals FlAsH and ReAsH could be split between two members of a protein partnership or two approximated regions of a protein while maintaining high affinity and brightness. Subsequently we explored the structural requirements of bipartite C4 display, and applied it to generate prototypes for encodable, fluorescent protein-free kinase sensors and p53 rescue agent sensors, as well as a strategy for the selective imaging of protein-protein complexes by electron microscopy. This renewal requests support for the continued development and application of bipartite tetracysteine display as well as a newly discovered orthogonal labeling strategy based on pro-fluorescent bis-boronic acids. We seek to achieve three major goals. The first (Aim 1) is a deeper, quantitative understanding of bipartite C4 display obtained through detailed kinetic, thermodynamic, structural, and photophysical experiments. We believe that the information obtained therein will inform and improve our ability to navigate and interpret the remaining experiments in this application and greatly facilitate the design of new bipartite-based experiments and sensors. The experiments in Aim 2 continue two projects from the previous funding period that possess the greatest potential impact. In Aim 2.1 we continue to develop encodable tyrosine kinase sensors based on bipartite C4 display, and apply them in collaboration to explore how Abl kinases coordinate cytoskeletal rearrangements in response to growth factors and adhesive cues. In Aim 2.2 we continue to develop sensors for molecules that stabilize oncogenic p53 variants, and applying them to identify new p53 small molecule chaperones. The experiments in Aim 3 explore the potential of bis-boronic acids as non-toxic, non-redox alternatives to biarsenicals for live cell imaging. We will evaluate a set of cyanine-based bis-boronic acids as brighter, more versatile alternatives for labeling serine-rich protein motifs, applying validated selection methods to identify optimal sequence tags. We will then build on these results to develop encodable RNA tags, and apply them in collaboration to visualize the mobility of Group II introns and trafficking of the hepatitis C virus RNA genome. PUBLIC HEALTH RELEVANCE: This proposal requests continued support to develop and apply bipartite tetracysteine display, and a novel orthogonal small molecule labeling strategy, to fluorescently image discrete protein conformations and protein assemblies in live cells. Detailed kinetic, thermodynamic, and photo-physical experiments will place the methodology on firm biophysical footing. Carefully chosen biological collaborations will showcase utility through applications to monitor Abl kinase, rescue destabilized p53, and monitor trafficking of the hepatitis C viral genome.